PPAR-γ regulates the effector function of human T helper 9 cells by promoting glycolysis

T helper 9 (TH9) cells promote allergic tissue inflammation and express the type 2 cytokines, IL-9 and IL-13, as well as the transcription factor, PPAR-γ. However, the functional role of PPAR-γ in human TH9 cells remains unknown. Here, we demonstrate that PPAR-γ drives activation-induced glycolysis, which, in turn, promotes the expression of IL-9, but not IL-13, in an mTORC1-dependent manner. In vitro and ex vivo experiments show that the PPAR-γ-mTORC1-IL-9 pathway is active in TH9 cells in human skin inflammation. Additionally, we find dynamic regulation of tissue glucose levels in acute allergic skin inflammation, suggesting that in situ glucose availability is linked to distinct immunological functions in vivo. Furthermore, paracrine IL-9 induces expression of the lactate transporter, MCT1, in TH cells and promotes their aerobic glycolysis and proliferative capacity. Altogether, our findings uncover a hitherto unknown relationship between PPAR-γ-dependent glucose metabolism and pathogenic effector functions in human TH9 cells.


REVIEWER COMMENTS
Reviewer #1 (expertise in immunometabolism, CD4+ T cell metabolism): In the paper by Bertschi and colleagues, the authors analyzed the role of peroxisome proliferator-                         glycolysis, which, in turn, specifically promotes the expression of IL-9 in an mTORC1-dependent manner. The authors corroborated their findings on skin samples from subjects with allergic contact dermatitis also showing that IL-9 induced the lactate transporter MCT1 expression, responsible of the increased glycolysis and proliferative capacity of Th9 cells.
                might represent novel therapeutic targets to modulate allergic contact dermatitis (ACD) as well as other Th2-driven diseases. Although the manuscript is quite clear and well structured, there are some pitfalls that partially limit the enthusiasm for its publication, as listed below: Major issues: 1) The first aspect concerns the methodological choice of using drugs to block different molecules (eg. Rapamycin, or GW9662, MCT inhibitor), with a pharmacological intervention. This approach can not exclude the possible off-target effects, also linked to the toxic effects of the used drugs. Therefore, it would be advisable to confirm the data also by inhibiting the expression of different molecules with specific silencing (ie. by shRNA) (genetic approach), in order to exclude side effects of the drugs, and more importantly, to be sure that the observed effects are specific and selective.                  this transcription factor (by pharmacological intervention). The authors should prove that, on the                                                               other pathways used for cellular energy support, such as mitochondrial respiration, glutaminolysis and fatty acid the oxidation. Is glycolysis the preferential pathway used by Th9 cells to produce energy? Do Th9 cells also use other intracellular metabolic pathways? All these aspects should be better investigated and discussed. 4) An aspect that deserves a deeper attention is the study of the molecular mechanism through               How does the regulation of its expression take place? is it a transcriptional regulation at the promoter level? through which mechanism does this process occur? Which factor is involved in this process? further details should be provided. 5) To link IL-9 with glycolysis and MCT1 expression, the authors should show the levels of IL-9 in the presence of MCT1 inhibitor and also, on the contrary, evaluate MCT1 expression when glycolysis is blocked. These experiments would provide a direct and univocal evidence of the role         Minor issues: 1) In addition to 2-DG, the authors should use and confirm their data also with other glycolysis inhibitors, acting on different molecules (ie. Lonidamine).
2) Immunofluorescence images of dermatological lesions from allergic contact dermatitis ( Figure  3F and H) should be compared to non-pathological conditions to better appreciate the difference, if any. Furthermore, those images should be quantified, in order to obtain a more objective estimation of the observed differences.
3) Cytofluorimetric histogram showing the CFSE staining ( Figure 2I) is very confusing. From the histogram it is very hard to observe a proper dilution of CFSE dye. How do the authors comment and interpret this data?
Since their characterization in 2008, Th9 cells have been shown to promote tissue inflammation in both mice and humans. Th9 cells shape disease course in multiple settings, including inflammatory and allergic diseases. While the transcription factor PU.1 has been identified as a major driver for IL-9 production for mouse and human CD4 T cells, most of the in vivo evidence showing the contribution of Th9 cells to disease stems from mouse studies. In this regard, the work presented in this manuscript outlines a very interesting signaling pathway contributing to CD4 T cell-derived IL-9 secretion in human skin inflammation. This study has many strengths: 1) It builds on previous mouse studies (notably PMID: 27317260) to extend to a human context the role of glycolysis and mTORC1 signaling in the secretion of IL-9 from Th9 cells.
2) It demonstrates the functional role of PPAR-g in driving glycolysis in human Th9 cells.
3) It mechanistically uncouples the secretion of IL-9 and IL-13 from effector Th9 cells. 4) It ascribes a novel role for IL-9 in enhancing the glycolytic capacity of IL-9R+ T cells Overall, these findings will further underscore the relevance of the Th9 cell subset in tissue inflammation. The data presented are for the most part convincing and logically build on the previous work from the authors published in Science Immunology.
While the data focusing on Th9 cell biology are straightforward, some sections of the manuscript need to be clarified to better integrate all relevant findings in the field. My concerns are as follows: 1) some sections of the manuscript are unclear and even conflicting with the message conveyed in the abstract. For instance, to begin their Materials and Methods section, the authors state that their goal was to study human pTH2 cells. Th9 cells are then described as a model to understand "pTh2" cells. Does this mean that the authors regard "pTh2" cells as a distinct CD4 T cell subset (as compared to conventional Th2 cells)? Would this mean that Th9 cells are not a distinct CD4 T cell subset? If the authors believe this, it is then somewhat odd that they rely in the present work on Th9 cells. Defining conditions to generate pTh2 cells in vitro would have been more appropriate. More importantly, while I understand that the authors are focusing on PPARg, it would be fair to discuss, and experimentally document in Figure 1, the RNA and protein levels of PU.1 considering previous literature (for instance: PMID: 20431622). This would also be important because in their Science Immunology paper, the authors actually reported higher PU.1 expression levels in Th9 cells as compared to Th2 cells at early time points during differentiation. 2) In line with the previous comment, while I understand the authors' willingness to discuss in detail in the introduction the concept they have written about (reference 2), I think additional balance would be beneficial. Except for my comment on PU.1, I concur that Figure 1 strongly suggests a very close proximity between pTh2 cells and Th9 cells. Surprisingly, despite the presented data, the authors do not discuss this here while they somehow addressed this in reference 2. Should pTH2 cells be renamed Th9 cells (or the opposite)? Or do the authors think that Th9 cells non-responsiveness to IL-33 is enough to discriminate pTh2 and Th9 cells, especially considering that some laboratories reported that Th9 cells respond to IL-33 (see for instance PMID: 29038366)? Presenting all perspectives would be very important to clarify the field and present a balanced view.
In summary, this is a very interesting study whose impact could be further enhanced by clearly underscoring the relevance of Th9 cells instead of representing them as a useful tool to study a larger group of inflammatory cells. This is actually what the authors have done in their abstract and graphical abstract, leading to very clear and convincing claims. Major comments: 1. The authors addressed the underlying molecular mechanisms by performing experiments of loss-of-function using inhibitors such as GW9662 and MCT1-i. The authors should perform the              confirm the molecular mechanisms that they found.
2. In the introduction part, the authors cited previous their-own review paper to introduce pathogenic Th2 cells. But there are other comprehensive review article regarding pathogenic Th2 cells such as Nakayama et al Annu Rev Immunol 2017. The concept of pathogenic Th2 cells was originally proposed by this group, and the paper should be included. Furthermore, in the discussion section, they mentioned the roles of IL-33-IL-33R pathway in both human and mouse pathogenic Th2 cells without the citation of appropriate original article, Endo et al Immunity 2015. They must include appropriate previous work.
3. The authors mentioned that mTORC1 activation induces IL-9 expression via HIF-1a in their Graphical abstract based on previous studies. But they did not present any data related to HIF-1a in the present study. Fig. 1G, the expression of IL9 seems to be significantly lower than that of IL5 and IL13. They should investigate the expression of IL9 expression by performing other experiments such as real-             upregulated in this experimental setting. 5. Since the authors have shown that TH9 cells specifically express high level of IL5 in Fig. 1C, they should address the production of IL-5 in Fig. 3A-C. 6. In Fig. 3C, the expression of PPARG should also be analyzed to investigate the activation of the    7. Regarding Fig. 4F and S4D, the authors should explain why they stained for CD3 or CD4 instead of the TH9 cell markers; CCR8 and CCR4 used in Fig. 2H seem to be more useful for markers to detect TH9 cells.

In
8. In Fig. 5C, immune-histological sample revealed that many CD3-negative IL-9R-expressing mononuclear cells were accumulated around CD3-positive T cells in the local tissues of allergic contact dermatitis. This result raised the possibility that IL-9 by TH9 cells may affect IL-9 receptor expressing CD3-negative cells, which consequently affect T cell function and glycolytic activity. To address this point, further experiments are required using in vivo primed TH9 clones, to measure the effects of IL-9 signal blockade using neutralizing antibodies against IL-9 or IL-9R blockade on glycolysis and MCT1 expression. 9. In Fig. 6D, how many hours after the start of the patch test were the glucose concentrations in the ACD model? The time course was clear in Fig. 1G, but not in this one. Also, were there any steroids or other products applied to the skin before or after the patch test?
Minor comments: 1. In Fig. 1E, the authors performed experiments using in vivo primed Th clones for the first time in this paper. To make it easier for readers to understand, they should add a brief explanation of the experimental system and describe it clearly in the Methods section. Fig. 4 B and C, in lines 175-176, the authors stated "Indeed, IL-9+ T cells were strongly enriched in the pS6+ cell population, whereas the proportion of IL-13+ T cells was similar in the pS6-and pS6+ populations," but in order to make this claim, the proportion of IL-9+ and IL-13+ cells should be shown.

Reviewer
In the paper by Bertschi and colleagues, the authors analyzed the role of peroxisome proliferator-activated receptor gamma (PPAR-) transcription factor in the regulation of the human T helper (Th)-9 cell effector functions. Specifically, they found that PPAR- controls activation-induced glycolysis, which, in turn, specifically promotes the expression of IL-9 in an mTORC1-dependent manner. The authors corroborated their findings on skin samples from subjects with allergic contact dermatitis also showing that IL-9 induced the lactate transporter MCT1 expression, responsible of the increased glycolysis and proliferative capacity of Th9 cells.
The paper is interesting and the topic is original as PPAR-, IL-9, and their downstream targets might represent novel therapeutic targets to modulate allergic contact dermatitis (ACD) as well as other Th2-driven diseases.
Although the manuscript is quite clear and well structured, there are some pitfalls that partially limit the enthusiasm for its publication, as listed below.
Major issues: 1.1 The first aspect concerns the methodological choice of using drugs to block different molecules (eg. Rapamycin, or GW9662, MCT inhibitor), with a pharmacological intervention. This approach cannot exclude the possible off-target effects, also linked to the toxic effects of the used drugs. Therefore, it would be advisable to confirm the data also by inhibiting the expression of different molecules with specific silencing (ie. by shRNA) (genetic approach), in order to exclude side effects of the drugs, and more importantly, to be sure that the observed effects are specific and selective.
Response: Thank you for this valuable suggestion. We have now included data with siRNA targeting i) PPARG, ii) RPTOR or iii) SLC16A1 that confirm our data obtained with the pharmacological inhibitors GW9662, rapamycin and BAY-8002, respectively.

i)
We show that silencing of PPARG leads to decreased glucose uptake, whereas fatty acid (FA) uptake is not affected. These results confirm our findings obtained with the PPAR- inhibitor GW9662 (new ii) We performed siRNA knockdown of the mTORC1 protein RPTOR. Efficiency and specificity of siRPTOR knockdown was validated by detection of reduced phosphorylation of S6. We then found that IL-9 levels are significantly reduced in knockdown cells, whereas IL-13 levels are unaffected, thus corroborating our results obtained with rapamycin (new Supplementary Fig. 4d, adapted text, results (line 197)).
iii) We show that successful silencing of SLC16A1 by siRNA suppresses extracellular lactate levels in cultured IL-9R + T H clones in the presence of IL-9, but not in cells transfected with control siRNA. This is consistent with our data obtained with the MCT1 inhibitor BAY-8002 (new Fig. 5m, adapted text, results (line 240)).
Changes to the revised manuscript: Response: Thank you for this suggestion. Since overexpression PPAR- is not sufficient to increase PPAR- signaling, as retinoid X receptor (RXR) as well as (the mostly unknown) endogenous ligands are also required, we decided to use the PPAR- agonist troglitazone (TGZ) in order to simulate PPAR- overexpression. However, unexpectedly, we did not detect higher IL-9 levels in presence of TGZ. Instead, we observed lower IL-9 levels, similar to the results obtained with the PPAR- antagonist GW9662. In parallel, we observed that S6 phosphorylation was reduced (new Supplementary Fig. 4e).
Previous studies have shown that PPAR- agonists such as TGZ activate AMPK 1 . We therefore hypothesized that AMPK activation by TGZ leads to mTORC1 inhibition that in turn decreases IL-9 levels. Indeed, using Western blot analysis, we show that TGZ leads to phosphorylation of AMPK and inactivation of mTORC1 (new Supplementary Fig. 4f). Moreover, the AMPK activator A769662 also reduces IL-9, but not IL-13 levels (new Supplementary Fig. 4g).
Although we did not show that overexpression of PPAR- leads to a reversal of the phenotype that we observed with PPAR- inhibition, the additional TGZ / AMPK activation data supports our hypothesis that IL-9 expression is regulated via the mTORC1 axis. These novel insights are included into the manuscript (new text, results (line 199-206)).
Changes to the revised manuscript: Since the PPAR- agonist Troglitazone (TGZ) unexpectedly reduced IL-9 expression in TH9 cells, we next investigated the mechanism by which this occurs. Interestingly, pS6 levels were reduced in presence of TGZ ( Supplementary Fig. 4e), suggesting that mTORC1 is inhibited. Previous studies have shown that PPAR- agonists, such as TGZ, activate AMP-activated protein kinase (AMPK) 1 . We therefore hypothesized that TGZ-mediated AMPK activation negatively regulates mTORC1, which in turn suppresses IL-9 expression. Indeed, Western blot analysis revealed that TGZ leads to phosphorylation of AMPK and mTORC1 inhibition ( Supplementary Fig. 4f). In addition, the AMPK activator A-769662 also reduced IL-9, but not IL-13 levels ( Supplementary Fig.  4g). Together, this data strongly supports our hypothesis that IL-9 expression is mTORC1-dependent." 1. 3 The authors focused their attention on the role of PPAR- in the induction and control of glycolysis.
However, it would be advisable also to show the effects of PPAR- modulation on the other pathways used for cellular energy support, such as mitochondrial respiration, glutaminolysis and fatty acid the oxidation.
Is glycolysis the preferential pathway used by Th9 cells to produce energy? Do Th9 cells also use other intracellular metabolic pathways? All these aspects should be better investigated and discussed.
Response: We thank the reviewer for this comment. As suggested, we performed additional experiments to study the effects of PPAR- inhibition on i) mitochondrial respiration, ii) fatty acid oxidation and iii) glutaminolysis.
i) To address the effects of two different PPAR- antagonists on mitochondrial respiration, we now show the data for oxygen consumption rate (OCR) measurements as measured by Seahorse flux analysis (new Supplementary Fig. 2c, d, adapted text, results (line 135-136; 157)). In accordance with the current literature (reviewed in 2 ) our data show that glycolysis is the dominant pathway engaged by activated T cells in high glucose environments, whereas mitochondrial respiration is only upregulated minimally. We therefore did not focus further on mitochondrial respiration because it is not the major metabolic pathway in the settings we studied (i.e. the conditions under which IL-9 is produced).
ii) Since PPAR- has been implicated in mediating fatty acid uptake in activated TH cells 3,4 , we examined fatty acid (FA) uptake in different T helper subsets. However, in contrast to glucose uptake, we found that TH9 cells did not exhibit higher FA uptake compared with TH1, and TH2 cells (new Supplementary   Fig. 2g). Further, PPAR- antagonism had no effect on FA uptake of in vitro-or in vivo-primed T H 9 cells -New text, results (line 180-183): "To investigate the contribution of FA metabolism to the regulation of cytokines in T H 9 cells, we analyzed IL-9 expression in response to FA inhibition. Neither did culturing of T H 9 clones in FA-free medium affect IL-9 or IL-13 expression ( Supplementary Fig. 3f), nor did inhibition of FA metabolism with etomoxir, an inhibitor of carnitine palmitoyltransferase-1 ( Supplementary Fig. 3g)." -New text, discussion (line 291-294): "PPAR- has previously been shown to be downstream of mTORC1 and to promote fatty acid uptake in activated T H cells 3 . In our study, however, mTORC1 activation was dependent on PPAR- activity and PPAR--antagonism had no effect of on FA uptake, neither in in vitro primed nor in in vivo primed T H 9 cells. The details of these discrepancies require further study." -New Supplementary Fig. 2j: Extracellular glutamine and glucose levels in TH9 cells in presence of GW9662.
-New text, results (line 154-155): "In addition, glutamine uptake of TH9 was not affected by PPAR-y inhibition, suggesting that glutaminolysis is not primarily regulated by PPAR-y in these cells ( Supplementary Fig. 2j)".

1.4
An aspect that deserves a deeper attention is the study of the molecular mechanism through which the PPAR- -mTOR-IL9 axis can regulate the expression of the lactate transporter MCT1. How does the regulation of its expression take place? is it a transcriptional regulation at the promoter level? through which mechanism does this process occur? Which factor is involved in this process? further details should be provided.
Response: Thank you for this comment. We have addressed this question in more detail and now show that the upregulation of the lactate transporter SLC16A1 by IL-9 is JAK3 dependent (new Fig. 5h). We suggest Response: We performed the suggested experiments. We now include data showing that IL-9 expression is significantly reduced in the presence of the MCT1 inhibitor BAY-8002, whereas IL-13 expression is unaffected (new Supplementary Fig. 5a). In addition, we now show that SLC16A1 expression is reduced in low glucose environments (new Supplementary Fig. 5b). These additional data are in line with the model proposed in our manuscript. The result section has been modified accordingly (line 240-243).
Changes to the revised manuscript: -New Supplementary Fig. 5a: Cytokine expression of TH9 cells in presence of the MCT1 inhibitor BAY-8002.
-New Supplementary Fig. 5b: SLC16A1 expression in low and high glucose environment before and after activation with CD3/CD2/CD28 -New text, results (line 240-243): "To link IL-9, glycolysis and MCT1 expression, we next investigated IL-9 levels in TH9 cells in presence of MCT1 inhibitor. We show that MCT1 inhibition reduces IL-9, but not IL-13 levels ( Supplementary Fig. 5a). On the contrary, low glucose environments, which in turn lead to reduced IL-9 levels, inhibited the induction of SLC16A1 (Supplementary Fig. 5b)." Minor issues: 1.6 In addition to 2-DG, the authors should use and confirm their data also with other glycolysis inhibitors, acting on different molecules (ie. Lonidamine).
Response: As suggested, we performed experiments with the glycolysis inhibitor lonidamine (LND). We show that LDN reduces IL-9 and IL-5 levels but not IL-13 expression, confirming our data obtained in low glucose and with 2-DG. Data are now included in the new Supplementary Fig. 3e and the text in the manuscript is adapted accordingly (line 174-175).
Changes to the revised manuscript: -New Supplementary Fig. 3e: Cytokine expression of TH9 cells in presence of LND.
-Adapted text, results (line 174-175): "In a next step, we hence inhibited glycolysis in TH9 cells using the glucose analog 2-deoxy-d-glucose (2-DG) and the aerobic glycolysis inhibitor lonidamine (LND) to investigate the effect on cytokine expression. In TH9 cells primed in vivo, 2-DG and LND inhibited the expression of IL-9 and IL-5 but not IL-13 ( Fig. 3d and Supplementary Fig. 3d, e)." 1.7 Immunofluorescence images of dermatological lesions from allergic contact dermatitis ( Figure 3F and H) should be compared to non-pathological conditions to better appreciate the difference, if any.
Furthermore, those images should be quantified, in order to obtain a more objective estimation of the observed differences.
Response: We thank the reviewer for this suggestion. We now show additional immunofluorescence images of normal skin (NS) and quantified CD4 + pS6 + , PPAR- + and PPAR- + pS6 + cells in ACD and NS, respectively.
The new data is included in in Fig. 4 f, h and the new Supplementary Fig. 4i, k. The text in the manuscript is adapted accordingly (line 208-210; 214-215).
Changes to the revised manuscript: -Adapted Fig. 4f and 4h: Quantification of CD4 + pS6 + and PPAR- + pS6 + cells in ACD and NS, respectively -New Supplementary Fig. 4i and k: Immunofluorescence images of NS stained for CD4 and pS6 -New Supplementary Fig. 4k: Immunofluorescence images of NS stained for PPAR- and pS6 as well as quantification of PPAR- + cells in ACD and NS.
-Adapted text, results (line 208-210): "…we performed immunofluorescence staining of normal skin (NS) and ACD skin samples and isolated T cells from such lesions. Double immunofluorescence revealed that CD3 + and CD4 + TH cells that express pS6 are significantly enriched in the infiltrate of ACD compared to NS ( Fig. 4f and Supplementary Fig. 4h,i)." -Adapted text, results (line 214-215): "Indeed, PPAR- + pS6 + double-positive lymphocytes were significantly increased in the dermis of ACD compared to NS ( Fig. 4h and Supplementary Fig. 4k)." Figure 2I) is very confusing. From the histogram it is very hard to observe a proper dilution of CFSE dye. How do the authors comment and interpret this data?

Cytofluorimetric histogram showing the CFSE staining (
Response: Thank you for this question. As T cells proliferate asymmetrically in our system, CFSE histograms usually display poorly resolved generations peaks 6 . For this particular experiment, we analyzed the MFI of CFSE to avoid arbitrary gating. However, to rule out a possible artifact in our experiments, we repeated this experiment using a BrdU assay to measure proliferation. We obtained the same results as for the CFSE experiment. The data is included into the new Supplementary Fig. 2f.
Changes to the revised manuscript: -New Supplementary Fig. 2f: BrdU assay of TH9 cells in presence of GW9662 and T0070907.

Reviewer
Since their characterization in 2008, Th9 cells have been shown to promote tissue inflammation in both mice and humans. Th9 cells shape disease course in multiple settings, including inflammatory and allergic diseases. While the transcription factor PU.1 has been identified as a major driver for IL-9 production for mouse and human CD4 T cells, most of the in vivo evidence showing the contribution of Th9 cells to disease stems from mouse studies.
In this regard, the work presented in this manuscript outlines a very interesting signaling pathway contributing to CD4 T cell-derived IL-9 secretion in human skin inflammation. This study has many strengths: 1) It builds on previous mouse studies (notably PMID: 27317260) to extend to a human context the role of glycolysis and mTORC1 signaling in the secretion of IL-9 from Th9 cells.
2) It demonstrates the functional role of PPAR-g in driving glycolysis in human Th9 cells.
4) It ascribes a novel role for IL-9 in enhancing the glycolytic capacity of IL-9R+ T cells Overall, these findings will further underscore the relevance of the Th9 cell subset in tissue inflammation. The data presented are for the most part convincing and logically build on the previous work from the authors published in Science Immunology.
Response: We thank the reviewer for this positive evaluation and the appreciation of our work.
While the data focusing on Th9 cell biology are straightforward, some sections of the manuscript need to be clarified to better integrate all relevant findings in the field.
My concerns are as follows: 2.1 Some sections of the manuscript are unclear and even conflicting with the message conveyed in the abstract. For instance, to begin their Materials and Methods section, the authors state that their goal was to study human pTH2 cells. Th9 cells are then described as a model to understand "pTh2" cells. Does this mean that the authors regard "pTh2" cells as a distinct CD4 T cell subset (as compared to conventional Th2 cells)? Would this mean that Th9 cells are not a distinct CD4 T cell subset? If the authors believe this, it is then somewhat odd that they rely in the present work on Th9 cells. Defining conditions to generate pTh2 cells in vitro would have been more appropriate.
Response: We thank the reviewer for raising this important question. Seminal work in mice and humans has indeed identified pathogenic TH2 cells (pTH2) as a distinct subpopulation within the TH2 cell subset. As such, they are regarded as distinct from conventional TH2 (cTH2) cells 7,8 . pTH2 cells differ from their cTH2 counterparts by their expression of specific cytokines (IL-5, IL-9), cytokine receptors (IL-9R, IL-17RB) and transcription factors (PPAR-). As shown in Fig. 1 of this manuscript, these characteristics are shared by TH9 cells. Given that TH9 cells also express key TH2 subset-defining properties 9 and in particular high levels of PPAR-, we propose here to use T H 9 cells as a model for studying the functional role of PPAR- in human T H cell biology.
However, we also observe clear differences between in vitro primed T H 9 cells and pT H 2 cells, such as their lack of IL-33R expression (discussed in the manuscript -line 278-280). These additional differentiation cues remain to be identified.
In summary, we believe that in vitro priming of TH9 cells recapitulates the key features of the pTH2 phenotype in type-2 driven diseases. We are grateful to the reviewer for pointing out that some sections in the manuscript, including the Study design in the Methods section, have been confusing for the reader. We have rephrased the suggested paragraphs accordingly (see below).
Changes to the revised manuscript: -Adapted text, introduction (line 93-94): "Here, we sought to investigate the mechanism by which PPAR- regulates the effector function of human TH9 cells, which share key characteristics with pTH2 cells." -Adapted text, results (line 106-108): "PPARG, IL5, IL17RB, and IL9R, which are hallmarks of pTH2 cells, were upregulated in TH9 cells as well as in all three pTH2 datasets, while IL9 was upregulated in two of the three (Fig. 1c and Supplementary Fig. 1a)." -Adapted text, results (line 112-113): "In summary, these findings strongly support our hypothesis that in vitro and in vivo primed TH9 cells share key similarities with pTH2 cells." -Adapted text, discussion (line 267-268): "Here, we used in vivo and in vitro primed TH9 cells, which represent a subpopulation of PPAR- + TH2 cells and share key characteristics with disease-associated pTH2 cells, to study the role of PPAR- in human TH cells".
-Adapted text, methods -study design (line 335-336): "The aim of this study was to investigate the mechanism by which PPAR- regulates the effector function of human TH9 cells, which share key characteristics with pTH2 cells. We used in vitro and in vivo primed PPAR- + TH9 cells and we performed RNA-seq analysis of activated TH9 clones upon treatment with the PPAR- antagonist GW9662." More importantly, while I understand that the authors are focusing on PPARg, it would be fair to discuss, and experimentally document in Figure 1, the RNA and protein levels of PU.1 considering previous literature (for instance: PMID: 20431622). This would also be important because in their Science Immunology paper, the authors actually reported higher PU.1 expression levels in Th9 cells as compared to Th2 cells at early time points during differentiation.
Response: This is an important remark and we agree that the previous literature on PU.1 requires discussion in our manuscript. In our Science Immunology article 9 we reported higher SPI1 mRNA levels (encoding PU.1) at day 3 during TH9 differentiation as compared to TH2 primed cells. At day 7, we observed no discernable difference in the expression of SPI1 levels using RT-qPCR. In the current study, we analyzed in vitro primed TH1, T H 2, T H 9 and iT REG cells at day 7 by RNAseq. In this data set, SPI1 is expressed at very low levels in T H 9 cells, albeit slightly but not significantly higher than in T H 2 cells. However, expression levels are very low in TH2 and TH9 cells when compared to iTREG cells (Adapted Supplementary Fig. 1a). In addition, SPI1 is not regularly detected in pT H 2 transcriptomes from human type 2 driven disease. Overall, we conclude that under the conditions used in the present study, SPI1 is neither specifically expressed by TH9 cells nor is it associated with the pathogenic phenotype in vivo. As suggested, we discuss these findings in the results section of the manuscript (line 108-110) and show the RNA levels of SPI1 in Supplementary Fig. 1a.
Changes to the revised manuscript: -Adapted Supplementary Fig. 1a: SPI1 expression levels of in vitro primed TH cell subsets at day 7 -New text, results (line 108-110): "In contrast, SPI1, encoding the transcription factor PU.1, previously shown to be associated with IL-9 expression 10,11 , was neither expressed in pTH2-specific transcriptomes, nor in TH9 cells (Supplementary Fig. 1a)." Response: We thank the reviewer for this comment. We now changed the manuscript and highlight that there is a very close proximity between pT H 2 and T H 9 cells and underscore the relevance of T H 9 cells (adapted text, results (line 106-108; 112-113)).
As written in comment 2.1, we observe clear discrepancies between TH9 and pTH2 cells, such as the expression of IL-33R, FAR3, and PTGDR2 (reviewed in 8 ), suggesting that certain differentiation cues for pTh2 cells are missing in TH9 cell differentiation. As long as these differentiations are ill defined, we think that it would be hasty to rename either pTH2 cells or TH9 cells.
The mentioned discrepancy we observe for the IL-33 responsiveness of TH9 cells and the previously published study 12 might arise from the different conditions used for in vitro priming. While we used IL-4 and TGF- to prime TH9 cells, Ramadan and colleagues additionally added IL-33 during priming to generate IL-33-responsive TH9 cells.
Changes to the revised manuscript: -Adapted text, results (line 106-108): "PPARG, IL5, IL17RB, and IL9R, which are hallmarks of pTH2 cells, were upregulated in TH9 cells as well as in all three pTH2 datasets, while IL9 was upregulated in two of the three ( Fig. 1c and Supplementary Fig. 1a)." -Adapted text, results (line 112-113): "In summary, these findings strongly support our hypothesis that in vitro and in vivo primed TH9 cells share key similarities with pTH2 cells." In summary, this is a very interesting study whose impact could be further enhanced by clearly underscoring the relevance of Th9 cells instead of representing them as a useful tool to study a larger group of inflammatory cells. This is actually what the authors have done in their abstract and graphical abstract, leading to very clear and convincing claims.

Reviewer
Bertschi et al. showed the pathological significance of PPAR in helper T cell 9 (TH9) cells by performing the functional analysis of PPAR using in vitro primed TH9 cells and TH9 cells derived from patients with allergic contact dermatitis.
First, the authors showed that TH9 cells expressed the core feature genes of pathogenic TH2 cells, including enhanced expression of PPAR-, IL-5, IL-9, and IL-9R. They showed that glucose uptake in TH9 cells was PPAR- dependent. Furthermore, they found that the production of IL-9 and IL-13 by TH9 cells was associated with the glycolytic activity. They also found that glucose and PPAR--dependent production of IL-9 in TH9 cells was regulated via mTORC1. Finally, they showed that IL-9 promotes aerobic glycolysis in IL-9R+ TH cells by inducing the lactate transporter MCT1.
This study demonstrates the importance of the PPAR--mTORC1-IL-9 axis in Th9 cells and is expected to aid in our understanding the type-2-driven skin inflammation. However, additional experiments are needed to support the authors' conclusions. Specific comments are described below.
Response: We thank the reviewer for this positive evaluation and the appreciation of our work. Response: Thank you very much for pointing this out. We agree that this could be misleading. We have now changed the graphical abstract accordingly. We have used different color for the HIF-1 axis and added the reference 18 to make it clear that this pathway was not investigated in the present study, but is based on previous literature. Since the layout of Nature Communications offers no opportunity to separately publish a Graphical Abstract, it is now included in Fig. 6e (adapted text, results (line 261-264)).
Changes to the revised manuscript: -Adapted Fig. 6e: Graphical abstract, different color for the HIF-1 axis, including reference -New text, results (line 261-264): "In addition, we found that PPAR- is a positive regulator of aerobic glycolysis in activated human T H 9 cells, which in turn, regulates the expression of IL-9 via mTORC1. Together, this suggests that PPAR- and IL-9 facilitate immunometabolic sensing of the tissue microenvironment (Fig. 6e)." Fig. 1G, the expression of IL9 seems to be significantly lower than that of IL5 and IL13. They should investigate the expression of IL9 expression by performing other experiments such as real-time quantitative PCR.

In
Response: Thank you for this comment. In Fig. 1g, we show the log2 fold change (FC) of the respective cytokine between the non-lesional control and 24 h, 48 h and 120 h after allergen application, respectively.
For IL5 and IL13 we therefore see a larger log2 FC compared with the non-lesional control. However, this analysis does not allow us to draw conclusions about the expression level. We therefore analyzed the counts of IL9, IL5 and IL13 expression form the RNAseq data and included this data in the manuscript (new Supplementary Fig. 1b).
We see that the IL9 and IL5 have similar expression levels, whereas the expression of IL13 is indeed higher.
This is congruent with what we observed in this study (i.e. Fig. 3b) as well as previously 9 . Moreover, we do not claim that IL9 and IL5 levels are higher compared to IL13 levels. Rather, we claim that the expression of IL9 and IL5 is higher in TH9 and pTH2 cells compared to conventional TH2 cells.
Changes to the revised manuscript: -New Supplementary Fig. 1b: Counts of IL9, IL5 and IL13 expression from RNAseq data The reviewer also wondered whether the expression of PPAR- was upregulated in this experimental setting.
Response: Thank you for raising this very important question. We have not included the PPARG data from the skin biopsy studies for two main reasons: i) Most importantly, PPARG is expressed in various skin cell types, including keratinocytes that vastly outnumber T H 9 cells in skin punch biopsies as used here (see Reference 9 and our Immunohistochemistry analysis for PPAR- below, left). We believe that the downregulation of PPARG we see in our experiment (see below, right) mostly represents the dedifferentiation of keratinocytes in allergic contact dermatitis rather than the expression of PPARG in TH9 cells 19 . We included this explanation in our manuscript (line 120-121).
ii) Secondly, since PPARG expression is very tightly controlled and precedes the expression of IL9, we believe that an earlier time point than 24 h would have been more appropriate to study PPARG levels in TH9 cells in skin. -Adapted text, results (line 169-171): "Subsequently, we performed RT-qPCR for IL9, IL13 and PPARG. Glucose uptake correlated with the expression of IL9 and PPARG, but not IL13 (Fig. 3c and Supplementary Fig. 3c)." 3.7 Regarding Fig. 4F and S4D, the authors should explain why they stained for CD3 or CD4 instead of the TH9 cell markers; CCR8 and CCR4 used in Fig. 2H seem to be more useful for markers to detect TH9 cells.
Response: Thank you for raising this point. Indeed, we have shown previously 9 and in this study that CCR4 and CCR8 are useful markers to identify TH9 cells by flow cytometry. We have been unable to establish specific and sensitive immunofluorescence stainings for CCR8 and CCR4 in ACD. However, on the one hand, we have previously established that virtually all IL-9 producing TH cells in human ACD express PPAR- 9 . On the other hand, new flow cytometry data show that virtually all IL-9 + T H cells isolated from ACD skin lesions express pS6, and thus have active mTORC1 signaling (new Supplementary Fig. 4j, line (210-211)). Thus, we have refrained from these immunofluorescence stainings in this study.
Changes to the revised manuscript: -New Supplementary Fig. 4j: Percentage of pS6and pS6 + cells in CD4 + IL-9 + TH cells isolated from ACD.
-New text, results (line 210-211): "Virtually all IL-9 + TH cells isolated from ACD show S6 phosphorylation, and thus have active mTORC1 signaling ( Supplementary Fig. 4j)." 3.8 In Fig. 5C, immune-histological sample revealed that many CD3-negative IL-9R-expressing mononuclear cells were accumulated around CD3-positive T cells in the local tissues of allergic contact dermatitis. This result raised the possibility that IL-9 by TH9 cells may affect IL-9 receptor expressing CD3-negative cells, which consequently affect T cell function and glycolytic activity. To address this point, further experiments are required using in vivo primed TH9 clones, to measure the effects of IL-9 signal blockade using neutralizing antibodies against IL-9 or IL-9R blockade on glycolysis and MCT1 expression.
Response: Thank you for this observation and suggestion. The aim of our study is to investigate the regulation of IL-9 by PPAR- in TH cells and the effects of paracrine IL-9 on TH cells. With this comment, the reviewer raises the question whether the observed effects of IL-9 on T cell function/glycolysis observed in our study could in fact be mediated via IL-9R + CD3-negative cells indirectly. This question is of course intriguing and worthy of further study.
However, we can exclude that the effects of IL-9 on TH cell function seen in our experiments requires the contribution of CD3-negative cells, as all our experiments ( Fig. 5e-m, Fig. 6a-c) were performed in the absence of any CD3-negative cells. We cannot exclude, however, whether in vivo, there is an additional indirect effect via CD3-negative cells.
To investigate the possibility that IL-9R + CD3-negative cells respond to IL-9 and this in turn may affect T cell function, we performed a transwell experiment. IL-9R + T cell clones were co-cultured either in the presence or absence of CD3-negative cells. As an indicator for glycolytic activity, proliferation in response to IL-9 was measured by CFSE dilution. As shown in the data below, we found no difference in the IL-9 response in presence or absence of CD3-negative cells. Rather, we observe slightly lower T cell proliferation when CD3negative cells are co-cultured and this is compensated by the addition of IL-9. We therefore conclude that the phenotype observed in our study is not induced via CD3-negative cells.

 
3.9 In Fig. 6D, how many hours after the start of the patch test were the glucose concentrations in the ACD model? The time course was clear in Fig. 1G, but not in this one. Also, were there any steroids or other products applied to the skin before or after the patch test?
Response  Table S3) 48 h post allergen application." -Adapted Supplementary Table 3: "biopsy collection post allergen application (hours)" -Adapted text, methods (line 455-456): "…and adjacent non-lesional skin biopsies were taken 48 h after allergen application. No steroids or other products were applied to the skin before or during the patch test." Minor comments: 3.10 In Fig. 1E, the authors performed experiments using in vivo primed Th clones for the first time in this paper.
To make it easier for readers to understand, they should add a brief explanation of the experimental system and describe it clearly in the Methods section.
Response: We thank the reviewer for pointing this out. To make it clearer to the reader, we have changed the main text in the result section and included a sentence on how in vivo primed TH9 clones were generated (line 110-111). We also rewrote the Methods section to explain in more detail how in vivo primed T cell clones were generated (line 362-367). In addition, we reference the gating strategy from our previous paper 9 in the method section (line 365).
Response: Thank you for this comment. We now included the basal acidification rate of the data shown in Fig 2c (new Supplementary Fig. 2b). In Fig. 2e, however, we did not use oligomycin, but performed an inrun activation experiment with CD3/CD2/CD28. Therefore, we did not include basal acidification rate for this experiment in the manuscript.
Changes to the revised manuscript: -New Supplementary Fig. 2b: Basal acidification rate of the data shown in Fig. 2c. 3.12 Fig. S3 seems to be missing.
Response: We named the supplementary figures after the respective main figures. In the initial submitted manuscript, we did not provide supplementary data for Fig. 3. However, in the new revised manuscript, we now show additional data for Fig. 3, which we present in the new Supplementary Fig. 3. Fig. 4 B and C, in lines 175-176, the authors stated "Indeed, IL-9+ T cells were strongly enriched in the pS6+ cell population, whereas the proportion of IL-13+ T cells was similar in the pS6-and pS6+

Regarding
populations," but in order to make this claim, the proportion of IL-9+ and IL-13+ cells should be shown.
Response: Thank you for pointing this out. In Fig. 4c, we actually display the ratio of IL-9 + and IL-13 + cells.
To make it clearer to the reader, we changed the figure legend and y-axis accordingly.
Changes to the revised manuscript: -Adapted y-axis and figure legend in Fig. 4c: Ratio (IL-9 + / IL-13 + ) highlighted in blue